The impact of replacing wheat flour with cellular legume powder on starch bioaccessibility, glycaemic response and bread roll quality: A double-blind randomised controlled trial in healthy participants.

The global rise in obesity and type 2 diabetes has generated significant interest in regulating the glycaemic impact of staple foods. Wheat breads (white or wholemeal) are popular staples, but have a high-glycaemic index, due to the highly digestible wheat starch. Reducing the glycaemic potency of white bread is challenging because the bread-making conditions are mostly conducive to starch gelatinisation. Cellular legume powders are a new source of type 1 resistant starch, where the starch is encapsulated by dietary fibre in the form of intact plant cell walls. The starch in these cell powders is less susceptible to gelatinisation and digestion than starch in conventional legume flours. However, legume cell resilience to baking conditions and the effects of this ingredient on glycaemic responses and product quality are unknown. Here we show that the integrity of cell wall fibre in chickpea powder was preserved on baking and this led to a ~40% reduction in in vivo glycaemic responses (iAUC120) to white bread rolls (~50 g available carbohydrate and 12 g wheat protein per serving) when 30% or 60% (w/w) of the wheat flour was replaced with intact cell powder. Significant reductions in glycaemic responses were achieved without adverse effects on bread texture, appearance or palatability. Starch digestibility analysis and microscopy confirmed the importance of cell integrity in attenuating glycaemic responses. Alternative processing methods that preserve cell integrity are a new, promising way to provide healthier low glycaemic staple foods; we anticipate that this will improve dietary options for diabetes care.

Hydrophobic Interactions Drive Binding between Vascular Endothelial Growth Factor-A (VEGFA) and Polyphenolic Inhibitors.

Some polyphenols have been shown to inhibit, at physiological levels, the VEGF-induced VEGF receptor-2 signaling that causes angiogenesis, allegedly by direct interaction with VEGF and reducing the binding to its receptor VEGFR2. Surface plasmon resonance was used to measure the parameters of binding between VEGF and polyphenols as well as the nature of the interactions by assessing the effect of physico-chemical changes in the solution. CD spectrometry was used to determine any change in the secondary structure of the protein upon binding. The kinetic parameters (ka, kd, and KD) that characterise the binding to VEGF were measured for both inhibitor and non-inhibitor polyphenolic molecules. The effect of changes in the physico-chemical conditions of the solution where the binding occurred indicated that the nature of the interactions between VEGF and EGCG was predominantly of a hydrophobic nature. CD studies suggested that a change in the secondary structure of the protein occurred upon binding. Direct interaction and binding between VEGF and polyphenol molecules acting as inhibitors of the signaling of VEGFR2 has been measured for the first time. The binding between VEGF and EGCG seemed to be based on hydrophobic interactions and caused a change in the secondary structure of the protein.

4-Week consumption of anthocyanin-rich blood orange juice does not affect LDL-cholesterol or other biomarkers of CVD risk and glycaemia compared with standard orange juice: a randomised controlled trial.

Elevated circulating cholesterol levels are a risk factor for CVD which is also associated with sub-optimal vascular function. There is emerging evidence that anthocyanins can cause beneficial cardio-protective effects by favourably modulating lipoprotein profiles. We compared the effects of blood orange juice which is rich in anthocyanins and blonde orange juice without anthocyanins on LDL-cholesterol and other biomarkers of CVD risk, vascular function and glycaemia. In all, forty-one participants (aged 25-84 years) with a waist circumference >94 cm (men) and >80 cm (women) completed a randomised, open label, two-arm cross-over trial. For 28 d participants ingested (i) 500 ml blood orange juice providing 50 mg anthocyanins/d and (ii) 500 ml blonde orange juice without anthocyanins. There was a minimum 3-week washout period between treatments. LDL-cholesterol and other biomarkers associated with CVD risk and glycaemia were assessed at the start and end of each treatment period. No significant differences were observed in total, HDL- and LDL-cholesterol, TAG, glucose, fructosamine, nitric oxide, C-reactive protein, aortic systolic blood pressure and diastolic blood pressure or carotid-femoral and brachial-ankle pulse wave velocity after 28 d ingestion of blood orange juice compared with standard orange juice. The lack of effect on LDL-cholesterol may be due to the modest concentration of anthocyanins in the blood orange juice.

Diet shapes the metabolite profile in the intact human ileum, which affects PYY release.

The human ileum contains a high density of enteroendocrine L-cells, which release the appetite-suppressing hormones glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in response to food intake. Recent evidence highlighted the potential role of food structures in PYY release, but the link between food structures, ileal metabolites, and appetite hormone release remains unclear owing to limited access to intact human ileum. In a randomized crossover trial (ISRCTN11327221; isrctn.com), we investigated the role of human ileum in GLP-1 and PYY release by giving healthy volunteers diets differing in fiber and food structure: high-fiber (intact or disrupted food structures) or low-fiber disrupted food structures. We used nasoenteric tubes to sample chyme from the intact distal ileum lumina of humans in the fasted state and every 60 min for 480 min postprandially. We demonstrate the highly dynamic, wide-ranging molecular environment of the ileum over time, with a substantial decrease in ileum bacterial numbers and bacterial metabolites after food intake. We also show that high-fiber diets, independent of food structure, increased PYY release compared with a low-fiber diet during 0 to 240 min postprandially. High-fiber diets also increased ileal stachyose, and a disrupted high-fiber diet increased certain ileal amino acids. Treatment of human ileal organoids with ileal fluids or an amino acid and stachyose mixture stimulated PYY expression in a similar profile to blood PYY concentrations, confirming the role of ileal metabolites in PYY release. Our study demonstrates the diet-induced changes over time in the metabolite environment of intact human ileum, which play a role in PYY release.

In vitro protein bioaccessibility and human serum amino acid responses to white bread enriched with intact plant cells.

The cell structure and low glycaemic benefits of pulses are compromised by conventional flour-milling. Cellular chickpea powders ('CCPs') are a new alternative to pulse flours. Here we investigated the in vitro bioaccessibility of essential amino acids ('EAAs') from CCP-enriched bread products and determined the effect of their consumption on serum amino acid responses in healthy humans (n = 20, randomised cross-over design). Breads were prepared with 0, 30 and 60 % of the wheat flour replaced by CCP (intact cells containing encapsulated protein). We found that significant proportion of EAAs from encapsulated protein became bioaccessible during in vitro duodenal digestion, and that in vivo serum EAA responses from healthy human participants were significantly higher following consumption of CCP-enriched breads. Furthermore, the EAA profile of in vitro digestion products were well-correlated with in vivo peak serum EAAs responses. We conclude that CCP-enrichment of wheat bread improved the amount and diversity of bioavailable EAAs.

Enhanced secretion of satiety-promoting gut hormones in healthy humans after consumption of white bread enriched with cellular chickpea flour: A randomized crossover study.

BACKGROUND: Dietary intake of pulses is associated with beneficial effects on body weight management and cardiometabolic health, but some of these effects are now known to depend on integrity of plant cells, which are usually disrupted by flour milling. Novel cellular flours preserve the intrinsic dietary fiber structure of whole pulses and provide a way to enrich preprocessed foods with encapsulated macronutrients. OBJECTIVES: This study aimed to determine the effects of replacing wheat flour with cellular chickpea flour on postprandial gut hormones, glucose, insulin, and satiety responses to white bread. METHODS: We conducted a double-blind randomized crossover study in which postprandial blood samples and scores were collected from healthy human participants (n = 20) after they consumed bread enriched with 0%, 30%, or 60% (wt/wt) cellular chickpea powder (CCP, 50 g total starch per serving). RESULTS: Bread type significantly affected postprandial glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) responses (time × treatment, P = 0.001 for both). The 60% CCP breads elicited significantly elevated and sustained release of these anorexigenic hormones [between 0% and 60% CPP-GLP-1: mean difference incremental area under the curve (iAUC), 3101 pM/min; 95% CI: 1891, 4310; P-adjusted < 0.001; PYY: mean difference iAUC, 3576 pM/min; 95% CI: 1024, 6128; P-adjusted = 0.006] and tended to increase fullness (time × treatment, P = 0.053). Moreover, bread type significantly influenced glycemia and insulinemia (time × treatment, P < 0.001, P = 0.006, and P = 0.001 for glucose, insulin, and C-peptide, respectively), with 30% CCP breads eliciting a >40% lower glucose iAUC (P-adjusted < 0.001) than the 0% CCP bread. Our in vitro studies revealed slow digestion of intact chickpea cells and provide a mechanistic explanation for the physiologic effects. CONCLUSIONS: The novel use of intact chickpea cells to replace refined flours in a white bread stimulates an anorexigenic gut hormone response and has potential to improve dietary strategies for prevention and treatment of cardiometabolic diseases. This study was registered at clinicaltrials.gov as NCT03994276.

An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes.

A set of mutant pea lines carrying induced mutations within the major seed-expressed starch-branching enzyme gene has been characterised at the molecular, chemical and agronomic levels. Eight of the induced mutations, three of which predicted a premature stop codon, were compared with the naturally occurring starch-branching enzyme mutation within the same genetic background. Starch, amylose and sugar measurements, coupled with analysis by ultra-high performance liquid chromatography-size exclusion chromatography of starches, identified a range of phenotypes which were grouped according to the nature of the mutation. Homology modelling of proteins supported the differences in phenotypes observed. Differences in field performance were evident for selected mutants, particularly in seed yield and mean seed weight traits for early compared with late spring sowings. The data show the potential of an allelic series of mutants at this locus for nutritional studies. CHEMICAL COMPOUNDS: starch, amylose, amylopectin, raffinose, stachyose, verbascose.

Characterization of the rate of thermally-induced aggregation of ß-lactoglobulin and its trehalose mixtures in the glass state.

The thermally induced aggregation of bovine ß-lactoglobulin in the glass state was studied both as the pure protein and in trehalose mixtures. Calorimetric measurements and size exclusion chromatography were used to characterize the glass state and aggregation, respectively. The initial aggregation rate was characterized by the initial rate of tetramer formation (ß-lactoglobulin dimerizes under the elution conditions), which showed Arrhenius temperature dependence with an activation energy of 95 kJ mol(-1) in the temperature range 60-100 °C. The trehalose addition slowed the aggregation in a way that depended exponentially on volume fraction, exp(-φ/φ*). The volume fraction characterizing the retardation of the reaction, φ*, was 0.173 of the order required to fill the interstices between close-packed protein molecules. In this system, trehalose was acting as an inert glass-forming diluent. In a general way, this supports the water substitute hypothesis and the understanding of the mechanisms underlying anhydrobiosis and protein stabilization technologies.

Effect of cooking, 24 h cold storage, microwave reheating, and particle size on in vitro starch digestibility of dry and fresh pasta.

The aim of this study was to investigate the effect of preparation method and particle size on digestion of starch in fresh and dry pasta types. Pasta samples were boiled, refrigerated and re-heated, with samples collected after each stage, and then prepared as small (2 mm) and large (5 mm) particles for subsequent starch digestibility testing and logarithm of slope analyses. There were significant main effects of particle size (F1,24 = 568.895, p < 0.001, ηp2 = 0.960) and processing treatment (F2,24 = 19.897, p < 0.001 ηp2 = 0.624) on starch digestibility overall, however the main effect of pasta type was not significant at the p < 0.05 level (F1,24 = 2.978, p = 0.097, ηp2 = 0.110). Particle size had the largest effect on digestibility, and the extent of starch digestion was at least 50% lower in samples prepared as large particles compared with small particles. The most digestible sample was the boiled fresh-type pasta prepared as small particles (C∞ = 57.9%) and cold storage alone and/or with subsequent re-heating significantly reduced the extent of digestion in this sample to ∼40.6%. In the dry pasta type, processing treatment had no significant effects on starch digestibility. The rate constant, k, was not significantly altered by processing treatment or pasta type (k = 0.0275 min-1, mean of all samples). These findings suggest that cold-storage and re-heating treatments have limited potential to impact on glycaemic responses and highlight the importance of masticated particle size as a potential rate-limiting factor in digestibility studies.

Monomeric Flavanols Are More Efficient Substrates for Gut Microbiota Conversion to Hydroxyphenyl-γ-Valerolactone Metabolites Than Oligomeric Procyanidins: A Randomized, Placebo-Controlled Human Intervention Trial.

SCOPE: The majority of ingested flavanols reach the colon where they are catabolized by the microbiota to form hydroxyphenyl-γ-valerolactones (HGVLs). It is not known if the HGVLs are catabolic products of monomeric (epi)catechins (EPC), oligomeric procyanidins (OPCs), or both. Using data from a randomized, double-blind, placebo-controlled crossover trial the relative contributions of catechins and OPC to the bioavailable pool of HGVLs are estimated. METHODS AND RESULTS: Participants ingested an apple extract once daily for 28 days that delivered the following: i) 70 mg EPC and 65 mg OPC (low dose EPC), ii) 140 mg EPC and 130 mg OPC (high dose EPC), iii) 6 mg EPC and 130 mg OPC (OPC), and iv) a placebo control. Urine is collected over a 24-h period before and after treatments. The median urinary excretion of HGVLs after ingestion of the high dose EPC is tenfold higher than that excreted after ingestion of the OPC that provided an equivalent dose of PC. Approximately 22% of catechins are converted to HGVLs in contrast to PC, for which there is limited conversion. CONCLUSION: Monomeric catechins are efficiently converted to derived HGVLs that are absorbed and excreted in human urine, whereas oligomeric PCs are much less efficiently converted.

A natural mutation in Pisum sativum L. (pea) alters starch assembly and improves glucose homeostasis in humans.

Elevated postprandial glucose (PPG) is a significant risk factor for non-communicable diseases globally. Currently, there is a limited understanding of how starch structures within a carbohydrate-rich food matrix interact with the gut luminal environment to control PPG. Here, we use pea seeds (Pisum sativum) and pea flour, derived from two near-identical pea genotypes (BC1/19RR and BC1/19rr) differing primarily in the type of starch accumulated, to explore the contribution of starch structure, food matrix and intestinal environment to PPG. Using stable isotope 13C-labelled pea seeds, coupled with synchronous gastric, duodenal and plasma sampling in vivo, we demonstrate that maintenance of cell structure and changes in starch morphology are closely related to lower glucose availability in the small intestine, resulting in acutely lower PPG and promotion of changes in the gut bacterial composition associated with long-term metabolic health improvements.

A single-enzyme system for starch digestibility screening and its relevance to understanding and predicting the glycaemic index of food products.

There is currently great interest in increasing provisions of healthier carbohydrate foods, particularly those that possess a low Glycaemic Index (GI) when measured in vivo. The metabolic response to many starch-rich foods is driven largely by differences in the rate and extent of starch amylolysis. Enzyme-kinetic parameters obtained from high-throughput in vitro amylolysis assays therefore have potential for rapid prediction of GI for starch-rich foods. The aim of this study was to evaluate the usefulness of a starch digestibility screening method and resulting enzyme-kinetic parameters in comparing and predicting the GI of a range of carbohydrate-rich foods. Starch-rich foods (n = 20) with GI ranging from 36 to 81 were digested by porcine pancreatic α-amylase for 90 min under a fixed enzyme-substrate ratio (4 U/10 mg starch) at 37 °C on a rotary mixer. Starch digestion progress was determined by quantification of reducing sugar concentration in aliquots collected throughout the incubation. Indices of starch digestibility (C20, C60, C90, HI, C∞, and k) were obtained and compared with GI values. Digestibility curves revealed differences in the starch amylolysis for the broad range of foods tested. In vitro starch digestibility indices were significantly correlated (p < 0.01) with GI, with the exception of the rate constant, k. Out of all the indices tested, C90 and C∞ were the most strongly correlated with in vivo rankings for GI of matched food products (Tb = 0.596, p < 0.001 and Tb = 0.599, p < 0.01, respectively), however the digestibility plots obtained for some of the more slowly digested foods were linear over 90 min meaning that C∞ and k could not be obtained from first order kinetic analysis. C90 was most strongly correlated with the absolute GI values (r = 0.724, p < 0.001). Overall starch digestibility profiles reflected differences in starch amylolysis for food with varying GI, and C90 provided the best indication of absolute and relative GI values across all product categories. The in vitro starch digestibility screening method shows potential for rapid prediction of GI values and is recommended for early stage food product development and for mechanistic studies.

Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) as an efficient tool for the rapid and highly informative characterisation of biopolymers.

Starch has a complex molecular structure, with properties dependent on the relative chain lengths and branching structure of its constituent molecules, which varies due to polymorphisms in starch biosynthetic genes, as well as environmental factors. Here we present the application of ultra-high performance size exclusion chromatography to the separation of starch chains from plant seeds. Several methods, have been used to analyse chain length distributions in starch, all with limitations in terms of analysis time, sample preparation and molecular weight range. Here we demonstrate that chain length distributions can be obtained with dramatically reduced analysis time using ultra-high performance size exclusion chromatography. The method may also show improvements in resolution of some fine structural features. Understanding links between starch fine structure and biosynthetic genes will allow bioengineering of starches with tailored properties. This technique may have application to the size separation and resolution of a range of biopolymers of value to the food, drink and pharmaceutical industries.

Comparative bio-accessibility, bioavailability and bioequivalence of quercetin, apigenin, glucoraphanin and carotenoids from freeze-dried vegetables incorporated into a baked snack versus minimally processed vegetables: Evidence from in vitro models and a human bioavailability study.

The aim was to incorporate vegetables containing the phytochemicals quercetin, apigenin, glucoraphanin and carotenoids into a processed potato-based snack and assess their bioaccessibility and bioavailability. Three different processing routes were tested for incorporation and retention of phytochemicals in snacks using individually quick frozen or freeze-dried vegetables. No significant differences in the uptake or transport of quercetin or apigenin between a vegetable mix or snacks were observed using the CaCo-2 transwell model. Simulated in vitro digestions predicted a substantial release of quercetin and apigenin, some release of glucoraphanin but none for carotenes from either the snack or equivalent steamed vegetables. In humans, there were no significant differences in the bioavailability of quercetin, apigenin or glucoraphanin from the snack or equivalent steamed vegetables. We have shown that significant quantities of freeze-dried vegetables can be incorporated into snacks with good retention of phytochemicals and with similar bioavailability to equivalent steamed vegetables.

Lack of acute or chronic effects of epicatechin-rich and procyanidin-rich apple extracts on blood pressure and cardiometabolic biomarkers in adults with moderately elevated blood pressure: a randomized, placebo-controlled crossover trial.

Background: The reported effects of flavanol-rich foods such as cocoa, dark chocolate, and apples on blood pressure and endothelial function may be due to the monomeric flavanols [mainly (-)-epicatechin (EC)], the oligomeric flavanols [procyanidins (PCs)], or other components. Reports of well-controlled intervention studies that test the effects of isolated oligomeric flavanols on biomarkers of cardiovascular health are lacking. Objective: We studied the acute and chronic effects of an EC-rich apple flavanol extract and isolated apple PCs on systolic blood pressure (BP) and other cardiometabolic biomarkers. Design: Forty-two healthy men and women with moderately elevated BP completed this randomized, double-blind, placebo-controlled, 4-arm crossover trial. Participants ingested a single dose of an apple flavanol extract (70 mg monomeric flavanols, 65 mg PCs), a double dose of this extract (140 mg monomeric flavanols, 130 mg PCs), an apple PC extract (130 mg PCs, 6.5 mg monomeric flavanols), or placebo capsules once daily for 4 wk, in random order. Biomarkers of cardiovascular disease risk and vascular function were measured before and 2 h after ingestion of the first dose and after the 4-wk intervention. Results: Compared with placebo, none of the isolated flavanol treatments significantly (P < 0.05) changed systolic or diastolic BP (peripheral and aortic), plasma nitric oxide (NO) reaction products, or measures of arterial stiffness (carotid femoral pulse-wave velocity, brachial-ankle pulse-wave velocity, or Augmentation Index) after 2 h or 4 wk of the intervention. There were no changes in plasma endogenous metabolite profiles or circulating NO; endothelin 1; total, HDL, or LDL cholesterol; triglycerides; fasting glucose; fructosamine; or insulin after 4 wk of the intervention. Conclusions: Our data suggest that, in isolation, neither monomeric flavanols nor PCs affect BP, blood lipid profiles, endothelial function, or glucose control in individuals with moderately elevated BP. The reported benefits of consuming flavanol-rich cocoa, chocolate, and apple products appear to be dependent on other components, which may work in combination with monomeric flavanols and PCs. This trial was registered at www.clinicaltrials.gov as NCT02013856.

A micro-reactor for preparing uniform molecularly imprinted polymer beads.

In this study, uniform spherical molecularly imprinted polymer beads were prepared via controlled suspension polymerization in a spiral-shaped microchannel using mineral oil and perfluorocarbon liquid as continuous phases. Monodisperse droplets containing the monomers, template, initiator, and porogenic solvent were introduced into the microchannel, and particles of uniform size were produced by subsequent UV polymerization, quickly and without wasting polymer materials. The droplet/particle size was varied by changing the flow conditions in the microfluidic device. The diameter of the resulting products typically had a coefficient of variation (CV) below 2%. The specific binding sites that were created during the imprinting process were analysed via radioligand binding analysis. The molecularly imprinted microspheres produced in the liquid perfluorocarbon continuous phase had a higher binding capacity compared with the particles produced in the mineral oil continuous phase, though it should be noted that the aim of this study was not to optimize or maximize imprinting performance, but rather to demonstrate broad applicability and compatibility with known MIP production methods. The successful imprinting against a model compound using two very different continuous phases (one requiring a surfactant to stabilize the droplets the other not) demonstrates the generality of this current simple approach.

Direct rapid synthesis of MIP beads in SPE cartridges.

Selecting optimal compositions for non-covalent molecularly imprinted polymers (MIPs) and screening for appropriate rebinding conditions necessitates synthesising a large number of polymers. This is extremely labour-intensive and usually results in very limited "optimisation" in studies of MIPs. Here, a new method is proposed for rapid synthesis of MIPs in a beaded form that can be used directly in many different performance evaluation studies. The method is based on synthesis of spherical particles by suspension polymerisation in liquid fluorocarbon [Mayes, A., Mosbach, K., 1996. Molecularly imprinted polymer beads: suspension polymerisation using a liquid perfluorocarbon as the dispersing phase. Anal. Chem. 68, 3769-3774]. The polymers were directly polymerised under UV light in solid phase extraction (SPE) cartridges, then washed and extracted in the same cartridges where they had been synthesised, resulting in a rapid and automatable process that requires no transfer or manipulation of the polymer particles. The particles were similar in terms of size, morphology and functional performance to particles obtained by suspension polymerisation in fluorocarbon solvent using a conventional reactor. In this initial study, 36 polymers were synthesised to study the effect of a variation in the type and amount of four different functional monomers, methacrylic acid (MAA), acrylic acid (AA), hydroxyethyl methacrylate (HEMA) and 2-vinylpyridine (2-VPy), for the imprinting of propranolol and morphine. The performance of polymers synthesised using MAA was as expected, but those synthesised with AA as functional monomer showed more surprising rebinding properties as a function of monomer to cross-linker ratios, demonstrating the potential value of pragmatic synthesis and screening approaches to polymer optimisation.

Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells.

UNLABELLED: Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003). Sub-group B (n = 9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009) and 35% (p = 0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p < 0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification. TRIAL REGISTRATION: ClinicalTrials.gov NCT01528644.

Evidence for an enhancing effect of alginate on iron availability in Caco-2 cells.

The potential use of alginates as a vehicle for water-soluble (bioavailable) iron for fortifying food products was examined using a Caco-2 cell model system. Cell monolayers were exposed to alginates with various mannuronic to guluronic acid ratios at three different concentrations, and cellular ferritin was measured as a surrogate marker of iron uptake into the cell. Ferritin concentrations were significantly higher when the cells were treated with ferric ammonium citrate and 0.5 and 1% w/v (but not 0.1%) alginate, but were unaffected by mannuronic/guluronic acid ratios. The enhancing effect of ascorbic acid was maintained with 0.1% alginate and significantly increased with 0.5 and 1% alginate, whereas the inhibitory effect of tannic acid was significantly reduced with 0.5% alginate. Alginate beads delivered available iron to Caco-2 cells, indicating that they are a promising vehicle for soluble iron with potential use in food fortification programs.

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices.

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state.